Impact of low lethal concentrations of buprofezin on biological traits and expression profile of chitin synthase 1 gene (CHS1) in melon aphid, Aphis gossypii

Buprofezin, a chitin synthesis inhibitor that can be used for the control of hemipteran pests, especially melon aphid, Aphis gossypii. The impact of low lethal concentrations of buprofezin on the biological parameters and expression profile of CHS1 gene were estimated for two successive generations of A. gossypii. The present result shows that the LC15 and LC30 of buprofezin significantly decreased the fecundity and longevity of both generations. Exposure of F0 individuals to both concentrations delay the developmental period in F1. Furthermore, the survival rate, intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R0) were reduced significantly in progeny generation at both concentrations. However, the reduction in gross reproductive rate (GRR) was observed only at LC30. Although, the mean generation time (T) prolonged substantially at LC30. Additionally, expression of the CHS1 gene was significantly increased in F0 adults. Significant increase in the relative abundance of CHS1 mRNA transcript was also observed at the juvenile and adult stages of F1 generation following exposure to LC15 and LC30. Therefore, our results show that buprofezin could affect the biological traits by diminishing the chitin contents owing to the inhibition of chitin synthase activity in the succeeding generation of melon aphid.


Results
toxicity of buprofezin on melon aphids. Buprofezin toxicity against A. gossypii was determined following 48 and 72 h exposure ( Table 1). The estimated value of LC 15  Sublethal effects of buprofezin on parental aphids (F 0 ). LC 15 and LC 30 concentrations of buprofezin have significant effects on parental A. gossypii following 72 h exposure. Both concentrations (LC 15 and LC 30 ) significantly decreased the longevity (F = 103.22; df = 2, 25; P < 0.001) and fecundity (F = 160.40; df = 2, 25; P < 0.001) of the exposed F 0 population. Furthermore, LC 30 concentration of buprofezin showed a stronger effect compared to LC 15 and the control ( Table 2).

Treatments
Slope ± SE a LC 15 mg L −1 (95% CL b ) LC 30 mg L −1 (95% CL b ) LC 50 mg L −1 (95% CL b ) χ 2 (df) c P www.nature.com/scientificreports www.nature.com/scientificreports/ individuals were significantly prolonged. Similarly the duration of 3 rd instar (F = 16.31; df = 2, 178; P < 0.001) and 4 th instar (F = 48.43; df = 2, 178; P < 0.001) also increased significantly at LC 30 of buprofezin. The total duration of pre-adult period (F = 273.70; df = 2, 178; P < 0.001) significantly increased in the offspring of F 0 generation after treated by both concentrations of buprofezin compared to the control. Paired bootstrap technique was applied to determine the transgenerational impact of buprofezin (LC 15 and LC 30 ) on population growth using TWOSEX MS chart program 54 . The population parameters of F 1 individuals, such as λ, r, R 0, and GRR were reduced at LC 30 concentration. Obvious increase was noted for the mean generation time (T) at LC 30 . However, no effects were observed for the LC 15 of buprofezin (Table 3). The age-stage specific survival rate (s xj ) curves indicated variations in the developmental rates occurring among juvenile stages. Moreover, overlapping between different immature stages were shown in control (Fig. 1A), LC 15 (Fig. 1B) and LC 30 concentration of buprofezin (Fig. 1C). The adult survival rates differed among the buprofezin treatments (LC 15 and LC 30 ) and the control. Furthermore, the declined survival rate of melon aphid adults for LC 30 concentration was recorded at the 12 th day (Fig. 1C) and the 16 th day was recorded for LC 15 concentration (Fig. 1B), while the decline survival rate of melon aphid adults for the control was recorded at the 23 rd day (Fig. 1A).
The age-specific survival rate (l x ), age-specific fecundity (m x ), and the age-specific maternity (l x m x ) for the treated and control A. gossypii are presented in Fig. 2. Compared to control treatment, the l x value for LC 15 and LC 30 concentrations of buprofezin declined more rapidly. The population started to decrease after 23 days in control ( Fig. 2A), whereas it declined after 16 days and 12 days in the LC 15 and LC 30 concentrations of buprofezin respectively (Fig. 2B,C).
The m x and l x m x values of the exposed A. gossypii were lower as compared to control (Fig. 2). The age-stage reproductive values (v xj ) of buprofezin treated adult aphids indicated that the v xj of LC 15 (Fig. 3B) and LC 30 (Fig. 3C) concentrations of buprofezin was lower in contrast to the control individual (Fig. 3A). The v xj value of LC 15 (6.80 at the age of the 6 th day) and LC 30 concentrations (4.9 at the age of the 7 th day) of buprofezin was lower compared to the control aphids (8 at the age of the 5 th day) (Fig. 3). Furthermore, the duration of F 1 aphid's reproduction was different after F 0 generation exposure to buprofezin (LC 15 and LC 30 ) compared to the control. The v xj value more than 4 was found for 17 days in the control group of melon aphid (Fig. 3A), while it was reported 14 and 7 days for LC 15 and LC 30 concentrations of buprofezin, respectively (Fig. 3B,C). The age-stage-specific life expectancy (e xj ) of buprofezin treated A. gossypii (LC 15 and LC 30 ) was lower as compared to the untreated control group (Fig. 4).

Sublethal effects of buprofezin on the expression profile of CHS1 gene in melon aphid.
Expression profile of CHS1 gene in melon aphids was evaluated by quantitative real-time PCR (qPCR) during F 0 adult and all developmental stages as well as newly emerged adult F 1 individuals. The results indicated that the mRNA level was up-regulated in F 0 adults and almost all stages of F 1 melon aphids at LC 15 and LC 30 of buprofezin, after the exposure of parent generation (F 0 ) for 72 h (Fig. 5). However, CHS1 gene was relatively highly expressed in F 0 adults treated with LC 30 (3.51-fold), while it was 2.59-fold increase for the case of LC 15 concentration of buprofezin. In the case of F 1 generation descending from the treated parent (F 0 ), CHS1 gene was constantly expressed in aphids from 1 st instar to the newly emerged adult aphid. The mRNA level of CHS1 was highly expressed in the 1 st instar (1.91-fold) and 2 nd instar nymph (1.44-fold) following exposure to buprofezin LC 30 . For LC 15 -treated group, the relative expression was  Table 3. Transgenerational effects of buprofezin on population parameters of the F 1 generation of A. gossypii whose parents (F 0 generation) were treated with LC 15 and LC 30 concentrations compared to untreated control. r: intrinsic rate of increase (d −1 ), λ: finite rate of increase (d −1 ), R 0 : net reproductive rate (offspring/individual), T: mean generation time (d), GRR: gross reproductive rate were calculated using 100,000 bootstraps resampling. Different letters within the same row show significant differences between control and buprofezin concentration groups (at the P < 0.05 level, paired bootstrap test using TWOSEX MS chart program).
www.nature.com/scientificreports www.nature.com/scientificreports/ 1.81-and 1.50-fold for the 1 st and 2 nd instar nymph respectively, compared to the control. The CHS1 gene was abundantly expressed 1.33-fold in 3 rd instar and 1.60-fold in the 4 th instar nymph after exposure to LC 30 , while they showed 1.24-and 1.53-fold increase for LC 15 concentration of buprofezin. In F 1 newly emerged adults, 2.50-and 1.78-fold abundance of the CHS1 were observed at LC 30 and LC 15 concentrations of buprofezin, respectively (Fig. 5). The transcriptional level of CHS1 gene increased 2.80-and 1.90-fold in parental aphids (F 0 ) following 48 h exposure to the LC 30 and LC 15 concentrations of buprofezin respectively ( Supplementary Fig. S1). While no effects were observed in the progeny generation (F 1 ). No significant increase was noted for the CHS1 gene transcription when melon aphids were treated to the two low lethal concentrations of buprofezin for 24 h (Supplementary Fig. S2).

Discussion
The impact of buprofezin on the biological traits and expression profile of chitin synthase 1 (CHS1) gene of melon aphid were investigated following exposure to low lethal concentrations of this pesticide. Sublethal effects of buprofezin, e.g. reduced longevity and fertility have been reported in various insect pests, e.g. Sogatella furcifera Horvath (Hemiptera: Delphacidae) 29,30 , Bemisia tabaci (Hemiptera: Aleyrodidae) 25 , Eretmocerus mundus Mercet www.nature.com/scientificreports www.nature.com/scientificreports/ (Hymenoptera: Aphelinidae) 25 , Encarsia inaron (Hymenoptera: Aphelinidae) 28 . In this study, the impact of buprofezin at low lethal concentrations were examined demographically among two subsequent generations of A. gossypii. The results showed a decrease in longevity and fecundity of A. gossypii at LC 15 , and even more markedly at LC 30 concentration of buprofezin in the progeny generation individuals. Similar effects were reported in the previous studies where the fecundity and longevity of S. furcifera females significantly reduced at sublethal doses of buprofezin 29,30 . The adult longevity and fecundity were decreased considerably when A. gossypii was treated to the LC 10 and LC 40 of cycloxaprid 55 . Additionally, low fertility has also been documented in A. gossypii 56 , B. brassicae 57 , and Diaphorina citri 58 at sublethal concentrations of imidacloprid. Buprofezin inhibits the prostaglandin biosynthesis in N. lugens when treated to the sublethal concentrations resulting in spawning suppression 59 . Similar results have also been reported for a low dose of pyriproxyfen in Aphis glycines Matsumura (Hemiptera: Aphididae) 60 , P. xylostella 41 and Choristoneura rosaceana 61 (Lepidoptera: Tortricidae) 62 . These studies suggested that low lethal or sublethal concentrations of insecticides, including IGRs, adversely affect the longevity as well as the fecundity of exposed insect pests, which can be widely diffused in various IPM programs. www.nature.com/scientificreports www.nature.com/scientificreports/ Transgenerational effects on the offspring of treated A. gossypii (F 0 ) were also found. The longevity and fecundity of F 1 individuals were decreased significantly at LC 15 and LC 30 concentrations, while the pre-adult period was increased. These effects are related to the reductions of F 1 demographical parameters. We had shown the data that the demographical parameters, e.g. r, λ, and R 0 were reduced significantly in F 1 generation when its parents (F 0 ) were subjected to LC 15 and LC 30 of buprofezin compared to the control; however, such negative impact on gross reproduction rate (GRR) was only evident at LC 30 concentration of buprofezin. Previously, similar effects were documented on the offspring of white-backed planthopper, Sogatella furcifera 29 , cotton aphid, A. gossypii 63 , and brown planthopper, Laodelphax striatellus 64 when subjected to the sublethal concentrations of buprofezin, sulfoxaflor, and thiamethoxam.
The analysis of the plotted curves for the s xj , l x , m x , l x m x, and e xj showed the adverse effects of buprofezin on the population growth parameters of A. gossypii. The v xj stated that the reproduction duration of melon aphids was negatively affected when exposed to low doses buprofezin. The pre-adult period and mean generation time (T) were increased due to different physical and chemical processes when treated with buprofezin. Similar effects www.nature.com/scientificreports www.nature.com/scientificreports/ at the demographical level have been presented in various other reports 29,30,65,66 . Previous reports suggested that exposure to sublethal concentrations of buprofezin can suppress the population growth of S. furcifera via impact on their survival and reproduction 29,30 . Additionally, adverse effects at the demographical level have been reported in melon aphid at 25 and 100 ppm of cucurbitacin B 65 . Moreover, sublethal concentrations of imidacloprid and pirimicarb decreased the longevity and population growth of A gossypii 67 . Soybean aphid also showed been reduced population growth when they were exposed to sublethal concentrations of imidacloprid 48 .
Chitin synthase 1 (CHS1) is crucial for the chitin synthesis 11 , which has been studied in various insects including soybean aphid, Aphis glycines, and brown citrus aphid, Toxoptera citricida 17,18 . In this study, the relative transcript level of CHS1 gene was up-regulated in the F 0 adult and in all nymphal stages of F 1 generation when exposed to LC 15 and LC 30 of buprofezin for 72 h. A previous study documented similar results for the white-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) when exposed to the LC 10 and LC 25 (0.10 and 0.28 mg/L) of buprofezin 30 . Additionally, the diflubenzuron exposure in insects including Anopheles quadrimaculatus Say (Diptera: Culicidae), Aphis glycines Matsumura (Hemiptera: Aphididae), Panonychus citri McGregor (Acari: Tetranychidae), and Toxoptera citricida Kirkaldy (Hemiptera: Aphididae) resulted in increased expression of CHS1 gene, which may be linked to www.nature.com/scientificreports www.nature.com/scientificreports/ increased mortality 17,18,68,69 . As the IGRs including buprofezin causes reduction in chitin content owing to the inhibition of chitin synthase activity in the exposed insect pests, which are translated into abortive molting, reduce longevity, decrease fecundity, and direct mortality 17,18,30,68,69 . In our investigations, the melon aphid population dynamics were reduced owing to the low lethal concentrations of buprofezin, suggesting their effectiveness against this insect pests. Moreover, the increasing abundance of CHS1 mRNA transcript may result from a regulatory feedback mechanism that compensates the CHS enzyme activity inhibited by buprofezin. The compensation mechanism that is proposed through overexpression of CHS1 gene translated into overproduction of the CHS1 protein. However, owing to the buprofezin exposure, the potential overproduction of CHS1 protein would be insufficient to maintain a vital level of CHS catalytic activity. Finally, the compensation mechanism failed to restore the enzymatic activity in the presence of buprofezin translated into reduced chitin production and causes the insect mortality.
In contrast to all these results, several studies documented no effect of insect growth regulator (e.g., diflubenzuron) on CHS1 gene expression in Drosophila melanogaster and Tribolium castaneum 70,71 . Therefore, future work needed to understand the biological significance of CHS1 gene comprehensively and as well as the relevant molecular mechanisms in the buprofezin exposed insects.
In conclusion, the LC 15 and LC 30 were used to understand the consequences of buprofezin on the biological traits and as well as their impact on the expression level of CHS1 gene in A. gosspii for over two successive generations. Results indicated a significant reduction of parental aphid's longevity and fertility when treated to the LC 15 and LC 30 of buprofezin. Moreover, both concentrations of buprofezin delay the aphid developmental stage and suppress the population growth of the progeny generation (F 1 ). Also, the CHS1 gene mRNA abundance was increased significantly at both concentrations following 72 h exposure. However, the effects observed from confined experimental scales may not translate into population effects under field contexts. Therefore, further investigation is necessary under field conditions to fully understand the potential of buprofezin's integration into an optimized IPM strategy to control this insect pest.

Materials and Methods
insects and insecticide. Melon aphid was originally collected from melon plants at Weifang District, Shandong Province, China. These insects were reared on fresh cucumber plants and were maintained under standard laboratory conditions with a temperature of 25 ± 1 °C, 70 ± 10% relative humidity (RH) and a 16:8 h light/dark photoperiod. Buprofezin with 97.4%, of active ingredient, was obtained from Jiangsu Anpon Electrochemical Co., Ltd. China. toxicity of buprofezin against Aphis gossypii. Toxicity of buprofezin was tested on A. gossypii using widely applied leaf dip bioassay procedure 48,66,72,73 . To ensure that all melon aphids were of same age and life instar, about 450 melon aphid adults were introduced on fresh cucumber plants. All adult aphids were removed after 24 hours while the offspring were allowed to grow on plants for eight days without any insecticide application. At this time, the newly-born nymphs passed all developmental stages and became apterous adults 65,66 .
The stock solution of buprofezin (active ingredient 97.4%) was prepared in acetone. The concentrations were further diluted with distilled water containing 0.05% (v/v) Triton X-100 to obtain six concentrations (8, 4, 2, 1, 0.5 and 0.25 mg L −1 ) for bioassays. Fresh leaf discs of cucumber plants were dipped for 15 s in the buprofezin solutions. After air drying, discs of cucumber plants were placed on agar bed (2%) in the 12-well tissue culture plate. Adult aphids were inoculated on the treated disc using a soft brush. The plates were covered with Parafilm (Chicago, USA). Each treatment has three replications, and 20-30 aphids per replicate were used in bioassay. Distilled water containing 0.05% (v/v) Triton X-100 was used as a control. All plates were placed in standard laboratory conditions with a temperature of 25 ± 1 °C, RH of 75% and a 16:8 h light/dark cycle (L:D). After 48 and 72 h exposures, aphid's mortality was checked. Aphids were considered dead if not show any movement after pushing gently 48,74 . Mortality of controlled aphids was less than 10%. PoloPlus 2.00 was used to determine the LC 15 , LC 30 and LC 50 of buprofezin. Figure 5. Relative expression levels of the chitin synthase 1 gene (CHS1) in F 0 adults and in all developmental stages along with newly emerged adult melon aphid of F 1 generation descending from the parent (F 0 ) exposed to the LC 15 and LC 30 concentrations of buprofezin for 72 h. The relative expression level is expressed as the mean (±SE) with the control as the calibrator. Different letters above the error bars indicate significant differences at P < 0.05 level (one-way ANOVA followed by Tukey HSD tests).
www.nature.com/scientificreports www.nature.com/scientificreports/ Sublethal effects of buprofezin on F 0 melon aphid. The life history traits of parental A. gossypii (F 0 ) were investigated following the previously described methods with slight modifications 65,66 . The stock solution of buprofezin was prepared using acetone. The tested concentrations of buprofezin (LC 15 and LC 30 ) was prepared in distilled water containing 0.05% triton X-100. The low lethal concentrations of buprofezin (LC 15 and LC 30 ) were selected to determine their impact on melon aphids, as most of the pesticides were degraded after initial application by various factors 34,75 . Insecticide exposure was carried out, as discussed above. After 72 h exposure, sixty live and healthy aphids were collected from buprofezin treatments (LC 15 , LC 30) and control. The apterous melon aphid adults collected from LC 15 , LC 30 and control were inoculated on fresh leaf discs individually 65,66 . Placed the treated discs on agar bed (2%) in the 12-well tissue culture plate. Parafilm (Chicago, USA) was used to cover the plate to prevent aphids escape. Fresh leaf discs were replaced throughout the experiment at every 3 rd day. All plates from buprofezin treatments (LC 15 , LC 30 ) and control were placed under laboratory conditions as mentioned above. Longevity, as well as fecundity of A. gossypii, were noted daily till death.
Transgenerational effects of buprofezin on F 1 Aphis gossypii. Impact of buprofezin at low lethal concentrations on the progeny generation (F 1 ) of melon aphids were evaluated using the same method and treatments as discussed previously. The newly-born nymphs were individually retained on each insecticide-free cucumber leaf disc, and they were used as F 1 generation of melon aphid. Sixty aphids were used for each of the treatment (LC 15 , LC 30 ) and control. Each aphid was considered as a single replication. The number of offspring were counted on a daily basis until the death of the adults. Impact of buprofezin on chitin synthase 1 gene expression at low lethal concentrations in melon aphid. Impact of buprofezin exposure on chitin synthase 1 gene expression in melon aphid was evaluated using the same experimental setup as described above. Survived healthy melon aphid adults were collected after 24, 48, and 72 h exposure and stored in −80 °C as F 0 generation. For F 1 generation, exposed aphids collected from LC 15 , LC 30 and control were transferred to new 20 mm diameter insecticide-free cucumber leaf discs. Aphids were collected at 4 developmental and newly emerged adult stages from both buprofezin treatments and control representing F 1 generation. Total RNA was isolated from the exposed A. gossypii using TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instruction. NAS-99 spectrophotometer (ACTGene) was used to analyze the RNA purity. Total RNA (1 μg) was used to synthesize the cDNA using the Data analysis. The LC 15 , LC 30 and LC 50 of buprofezin were analyzed using a log-probit model 78 as commonly used in various studies 42,48 . The demographical (r, λ, R 0 , T, and GRR) and basic life table parameters (s xj , v xj , l x , m x , l x m x, and e xj ) were calculated using TWOSEX-MSChart program 54,79,80 . The intrinsic rate of increase (r) is classified as the population growth rate when the time advances infinity and population attains the stable age-stage distribution. The population will rise at the rate of e r per time unit. It was calculated using eq. 1: x r x x x 0 ( 1) The finite rate of increase (λ) is defined as the population growth rate as the time approaches infinity and population attains the stable age stage distribution. It was calculated using Eq. 2:

Primer name
Sequence (5′-3′) Application Table 4. Primer sequences for chitin synthase 1 (CHS1) and internal control genes used to determine the expression profile in A. gossypii following exposure to buprofezin.
www.nature.com/scientificreports www.nature.com/scientificreports/ The net reproductive rate (R 0 ) is classified as the total fecundity produce by a common insect pest during the whole life. It was calculated using Eq. 3: The mean generation time (T) is the duration of time that is needed by a population to enhance to R 0 -fold of its size as time advances infinity and the population calms down to a persistent age-stage distribution. It was measured using Eq. 4: The gross reproduction rate (GRR) was measured using Eq. 5: The age-specific survival rate (l x ) was measured using Eq. 6: where m is the number of stages. Age-specific fecundity (m x ) was measured through Eq. 7: Where s′ ij is the probability of an insect of age x and stage y will endure to age i and stage j. The age-stage reproductive value (v xj ) is classified as the expectation of an insect of age x and stage y to the future offspring. It was measured using Eq. 9: Population parameters were calculated and compared through paired bootstrap test 81 using TWOSEX-MSChart with 100,000 replicates 53,82 . The results related to fecundity, longevity, developmental periods and CHS1 expression of A. gossypii were calculated by One-way analysis of variance (ANOVA) with Tukey post hoc test (P < 0.05) (IBM, SPSS Statistics).

Data Availability
All data generated and analysed during this study are included in this published article (and its Supplementary Files). Supplementary www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/